Fundamental to understanding how flux through a metabolic pathway is controlled by a regulatory enzyme is knowledge of the mechanism of action of that enzyme. It is the objective of the proposed research to determine the enzymatic mechanisms by which three regulatory enzymes, i.e. acetyl-CoA carboxylase, ribulose diphosphate carboxylase, and HMG-CoA synthase, catalyze their respective reactions. Among the major metabolic pathways regulated by these enzymes are fatty acid synthesis, cholesterogenesis, and ketogenesis. Particular emphasis will be given to assessing the roles of reactive enzyme-bound intermediates tentatively identified during the preceding grant period. These include: 1) a carbonic-phosphoric anhydride intermediate in the case of acetyl-CoA carboxylase and other biotin-containing carboxylases, 2) a 2-carboxy-3-keto ribitol diphosphate intermediate in the case of ribulose diphosphate carboxylase, and 3) an S-acetyl- (and perhaps S-HMG-CoA acyl-) cysteinyl enzyme intermediate in the case of HMG-CoA synthase. Since all three enzymes are multisubunit structures, two of which are known to be composed of non-identical subunits, an attempt will be made to assign specific catalytic (or regulatory) functions to each component subunit.